Genomic imprinting is a parental origin-specific gene silencing that leads to differential expression of the two alleles of a gene in mammalian cells. Imprinting has attracted intense interest for several reasons: (i) Imprinting is by definition reversible and may be regulated over a large genomic domain (1). (ii) Imprinted genes and the imprinting mechanism itself are important in human birth defects and cancer (2). (iii) It has been suggested that imprinting cannot be reprogrammed without passage through the germline and thus constitutes a barrier to human embryonic stem cell transplantation (3).
Experimental studies of the timing and mechanism of genomic imprinting have been hampered by the fact that imprinting requires passage through the germline, analysis of which poses a difficult experimental target. Thus, there is a need in the art for an experimental model system which allows direct examination of allele-specific gene silencing in the dynamic process of genomic imprinting.
DNA methylation is central to many mammalian processes including embryonal development, X-inactivation, genomic imprinting, regulation of gene expression, and host defense against parasites, as well as abnormal processes such as carcinogenesis, fragile site expression, and cytosine to thymine transition mutations. DNA methylation in mammals is achieved by the transfer of a methyl group from S-adenosyl-methionine to the C5 position of cytosine. This reaction is catalyzed by DNA methyltransferases and is specific to cytosines in CpG dinucleotides. 70% of all cytosines in CpG dinucleotides in the human genome are methylated and prone to deamination, resulting in a cytosine to thymine transition. This process leads to an overall reduction in the frequency of guanine and cytosine to about 40% of all nucleotides and a further reduction in the frequency of CpG dinucleotides to about a quarter of their expected frequency (35). The exception to this rule are CpG islands, that were first identified as HpaII tiny fragments (36), later to be defined as sequences of 1-2 kb with a GC content of above 50% and a frequency of CpG dinucleotides greater than 0.6 of their expected frequency (37). CpG islands have been estimated to constitute 1-2% of the mammalian genome (38), and are found around the promoters of all housekeeping genes, as well as in a less conserved position in 40% of tissue specific genes (39). The persistence of CpG dinucleotides in CpG islands is largely attributed to a general lack of methylation, regardless of expression status (reviewed in ref. 40).
The two exceptions to the rule of CpG islands being unmethylated in normal cells, are on the inactive X chromosome (41) and in association with imprinted genes (42, 43). Genomic imprinting is the differential expression of the two parental alleles of a gene, and most imprinted genes are associated with at least one CpG island methylated uniquely on a specific parental chromosome (42). In addition, aberrant methylation of CpG islands has been observed in tumors and cultured cells, and it is thought to be a mechanism to silence tumor suppressor genes (44, 45).
Numerous approaches have been used to identify CpG islands that are differentially methylated in specific cell types, such as tumor-normal pairs for cancer-related methylation changes (46-48), or differential parental origin for imprinted genes (49-50). However, there was only one report of a systematic effort to identify CpG islands throughout the genome that might be normally methylated (51) using a methyl-CPG binding column. However, the resulting sequences were mainly dispersed repeats, ribosomal DNA and other repeated sequences with no characterization of unique, methylated CpG island.
There is a need in the art for identification of unique, methylated CpG islands so that imprinted genes can be identified.